Method to select and transfect cell subpopulations

ABSTRACT

A method for transfecting and separating cells is disclosed. The method comprises preparing magnetic particles coated with genetic material and a cell-specific ligand, and using the particles to transfect target cells. The target cells may then be separated from the non-target cells by using a magnetic field.

This application is the national stage of International Application No.PCT/US97/13523, which claims the benefit of U.S. Provisional ApplicationNo. 60/023,063, filed Aug. 2, 1996.

BACKGROUND OF THE INVENTION

The infection or transfection of cells by infectious viruses orretroviruses has been known for many years. Many methods for improvingthe rate of transfection have been used. Calcium chloride treatment ofthe cells has been used to allow naked DNA to pass through the cellularmembrane. Improved vectors have been developed for increasing the levelof infection. The use of lipids or liposomes to deliver the geneticmaterial have also been used with some amount of success.

Particle mediated delivery has been used to transform numerous types ofcells with a gold microparticle mediated delivery of DNA. The techniquehas been described in numerous publications and has advanced enough tomerit a product which has been marketed by BioRad under the nameBiolistic PDS. The use of this and similar systems are described in U.S.Pat. Nos. 4,945,050; 5,015,580; and 5,120,657 and international patentapplications WO 95/29703 and WO 93/17706. An improvement upon theparticle mediated delivery was made by Palsson, as disclosed in U.S.Pat. No. 5,534,423. The Palsson method includes loading a vector and thecells to be transformed into a centrifuge apparatus and spinning it tobring the vectors into contact with the cells. A spinnable disk and anelectrolytic system are also envisioned.

SUMMARY OF THE INVENTION

The instant invention provides a method for transfection of target cellsby which the transfection efficiency of vectors and target cells isimproved by bringing the target cell and the vector in contact. This isachieved by immobilization of both the vector containing the relevantgenetic material and the ligand specific for the target cells on aparticle. When such particle is now incubated with a test sampleconsisting of a cell mixture containing the target cells, the ligandspecific for the target cells will bind to the target cells and thusbring the vector in contact with the target cells. The target cells cannow be separated from the non target cells by isolation of theparticle-cell construct, thus achieving both enrichment of target cellsand transfection of the target cells.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic diagram of the separation and transfection of atarget cell with a particle associated with a vector.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “transfection” means the introduction of DNA,RNA, other genetic material, protein or organelle into a target cell.

As used herein, the term “vector” means any particle capable oftransfecting a target cell. Vectors known to the art include, forexample, viruses, spheroplasts or liposomes containing genes, and freenucleic acids containing genes, such as plasmids or nucleic acidfragments. Viruses useful in the methods of this invention includeretroviruses (such as murine amphotropic virus), baculovirus, SV40-typeviruses, polyoma viruses, adenoviruses, Epstein-Barr viruses, herpessimplex virus, vaccinia viruses and papilloma viruses. One can readilyemploy other vectors not named but known to the art. The methods of thisinvention are particularly useful for increasing rates of infection byvectors having half-lives of less than 24 hours, such as the murineamphotropic virus and baculovirus. However, they are also useful forincreasing rates of infection for vectors having longer half-lives.Adsorption of vectors by cells depends, in part, on concentration of thevector in solution. By imparting directed motion to the vectors in thedirection of the cells, the methods of this invention effectivelyincrease the concentration of the vectors in the vicinity of the cells,resulting in more adsorption and infection.

As used herein, the term “target cell” refers to cells capable of beinginfected by a vector. Target cells may originate from any unicellularorganism, plant, or animal, and include those cells of human origin.Cells useful for gene therapy are particularly useful in this invention.They include, for example, bone marrow cells, lymphocytes, fibroblasts,keratinocytes, hepatocytes, endothelial cells, neurons, muscle cells,epithelial cells, hematopoietic stem cells. This invention furthercontemplates use of cells taken from a patient or subject with theintent of infecting those cells and re-introducing them into the patientor subject.

As used herein, the term “particle” refers to any solid micro-particlewith a diameter between 30 nm to 5 microns. These particles may be madeof any type of natural or synthetic material and are optionally coatedwith a natural or synthetic polymer. Microparticles of the inventioninclude polystyrene, latex, and metal oxides. Preferred are particlesbetween the sizes of about 3 nm and 200 nm. Particularly preferred aremagnetic microparticles particles between the sizes of about 30 nm and200 nm.

The term “magnetic microparticle” refers to a material which may or maynot be permanently magnetic, which also may be paramagnetic orsuper-paramagnetic but which in all cases exhibits a response in amagnetic field, i.e., is magnetically responsive. Many techniques havebeen suggested in the prior art for the preparation of such magneticparticles or organo-magnetic materials, including U.S. Pat. Nos.5,512,332; 4,795,698; 4,230,685; 3,970,518; 4,554,088; 3,970,518 and4,018,886. European patent number 0 489 119 also teaches the preparationof resuspendable coated particles.

The magnetic particles of the current invention are preferably betweenthe sizes of 30 nm and 200 nm and are optionally coated with a naturalor synthetic polymer. Associated with this magnetic particle is thevector used to transfect the cell of interest. The magnetic particlescould be coated with or specifically linked to DNA, RNA, a virus, aretrovirus, a prion, or other infectious material as described above.Also associated with the magnetic particle is an immunospecific ligand.The ligand can be linked through a covalent or non-covalent bond to afunctionality on the surface of the magnetic particle, or the ligand maybe used to coat the particle. In one embodiment, the ligand coatedmagnetic particle and the vector associated magnetic particle are notthe same.

One embodiment of the instant invention is schematically represented byFIG. 1. In FIG. 1, the target cell 6 will be approx. 5-50 mm indiameter. Receptor 5 is located on the surface of the target cell andspecifically interacts with immunospecific ligand 3, which is coatedupon particle 4, which has a diameter of 30-200 nm. Also associated withparticle 4 through bond 2 is vector 1, which will have a size of approx.1-100 nm. In this FIGURE, the target cell and the particle associatedwith the vector have already been brought into contact, allowing theimmunospecific ligand 3 to react with the receptor 5 on the surface ofthe target cell 6, thus forming a particle-cell construct. Uponincubation of the particle cell construct under the appropriateconditions of temperature and culture media, infection of the targetcell by the vector should result in the transfer of genetic material tothe cell and thus to transfection of the target cell. Optionally, bond 2may be broken after formation of the particle-cell construct to furtherfacilitate infection of the target cell by the vector, for example,using the release techniques described in commonly owned, co-pendingU.S. application Ser. No. 08/395,967, the entire disclosure of which isincorporated by referenced herein.

When desired, isolation of the particle-cell construct could be throughthe use of a column, coated surface, or any other means of particlemanipulation known in the art. The method of the instant inventionbrings the target cell and the vector desired to transfect said cellinto close proximity, thus improving the chances that the vector will infact transfect the target cell. In other words, the local concentrationof vector and target cell are increased.

In a preferred embodiment, a magnetic microparticle is coated withimmunospecific ligand and associated with a vector. Once the target celland the magnetic particle have been brought into contact, a magneticallyresponsive cell construct is formed. When this cell construct comesunder the influence of a magnetic field gradient, either by theplacement of the test sample containing the cell construct into amagnetic device, the generation of a magnetic field in the containerwhich holds the test sample or the flowing of the test sample through aflow-through device, one section of which includes a magnetic fieldgradient, the cell construct will respond to the field gradient, movingto the region of highest field, thus separating from the remainder ofthe test sample. Optionally, the remaining test sample which is notmagnetically responsive can be removed. This magnetic embodiment of theinstant invention provides for the separation of the target cell fromthe test sample, thus limiting the amount of manipulation necessary inthe transfection protocol, which will further increase cell viabilityand therefore transfection efficiency.

Although FIG. 1 depicts the direct specific interaction of ligand 3 withreceptor 5, it should be apparent to one skilled in the art, that anindirect specific interaction is also possible. For example abiotinylated monoclonal antibody which is specific to the cell surfacereceptor may be incubated with the test sample which contains the targetcell. In this embodiment a magnetic particle coated with avidin orstreptavidin could be used to select the target cell. It should also beobvious to one skilled in the art that any type of ligand would beuseful in order to practice the teaching of the instant invention. Forexample, a monoclonal antibody would provide a specific interaction withthe cell, but also envisioned is the use of single chain antibodies,polypeptides selected from a library of peptides, or polyclonalantibodies.

In another embodiment of the instant invention, a plurality of magneticparticles are employed. One set of magnetic particles can be coated withligand which specifically interacts with the target cell, eitherdirectly or indirectly, as described above. A second set of magneticparticles may be associated with the vector containing the geneticmaterial to transfect the cell. After incubation of the test sample withthe ligand-coated magnetic particles, the vector-associated magneticparticles could be added and the magnetic field gradient generated. Upongeneration of the field gradient in the test sample, the target cell andthe vector would come into close proximity and the opportunity for thevector to transfect the cell would be increased. Optionally, thevector-associated magnetic particles could be added to the target cellsafter said cells had been collected under the influence of the magneticfield gradient. When the vector associated magnetic particles come intothe field gradient, they will accelerate toward the target cell,increasing the probability of transfection.

EXAMPLE Enrichment and Transfection of Hematopoietic Progenitor Cells

Gene transfer technologies are of interest in view of their potential tocorrect a large variety of somatic cell defects or to make thetransduced cells resistant to treatments which are otherwise detrimentalfor these cells. A procedure commonly used to enrich for hematopoieticprogenitor cells is to select cells expressing the CD34 antigen frombone marrow or peripheral blood. This is for example achieved byimmobilization of monoclonal antibodies specific for CD34 on magneticparticles. After incubation of the cell suspension with theimmunomagnetic particles, the cell suspension is placed in or passedthrough a magnetic separator and the cells labeled with immunomagneticparticles are separated from the cell mixture. The CD34+ enriched cellsare then exposed to supernatants containing for instance retroviralvectors. This transfection process is inefficient and generally resultsin a low percentage of transfected target cells. A means to increasethis efficiency is to increase the movement of the particles towards thetarget cells such that the random Brownian motion is overcome. In thepresent invention the movement of the vector towards the target cell isachieved by immobilizing both the vector (retrovirus containing thegenetic information) and the ligand (anti-CD34) on a magnetic particle.After an incubation period of a bodily fluid containing the target cellswith the anti-CD34 and retrovirus labeled magnetic particle, the fluidis exposed to a magnetic field. The target cells labeled with themagnetic particles are then concentrated at the surface with the highestmagnetic gradient which further increases the specific contact betweentarget cells and vectors. The target cells are thus separated from theheterogeneous mixture and simultaneously brought in contact with theretrovirus permitting a more efficient transfection.

I claim:
 1. A method for transfecting a target cell with DNA andmagnetically selecting the resulting cell from a test sample, comprisingthe steps of: providing magnetic particles coated with a ligand whichspecifically binds, either directly or indirectly, to said target cell,said magnetic particles being associated with a vector containing saidDNA; contacting a test sample containing said target cell with saidmagnetic particles under conditions forming a magnetically responsiveparticle-cell complex and effecting transfection of said target cellwith said DNA via said vector; and generating in the test sample amagnetic field gradient of sufficiently high intensity to separatemagnetically the transfected target cells from said test sample.